Targeted Multi-Spectral Dual Axes Confocal Imaging of In Vivo Molecular Expression The broad, long-term objective of this BRP is to develop a miniature intra-vital imaging instrument to study molecular mechanisms of cancer biology in the epithelium of live animals. This novel optical design uses off-axis illumination and collection of light to produce superior dynamic range for collecting vertical cross-sectional fluorescence images. We will assemble an inter-disciplinary team of investigators from the College of Engineering and the School of Medicine at the University of Michigan. The specific aims are 1) to develop a scanning mechanism to achieve real time, multi- spectral vertical cross-sectional imaging, 2) to select affinity peptides that bind to dysplasia in colonic mucosal epithelium and to CXCR7 in breast vascular endothelium, and 3) to validate imaging performance in live animal models. The dual axes confocal architecture uses the overlapping focus of two separate low numerical aperture objectives to achieve sub-cellular resolution and long working distance. Post-objective scanning provides a large field-of-view as well as scalability and miniaturization of the optics, and a replicated parabolic mirror directs collimated, multi-spectral beams to a common focus. This unique design can view the epithelial differentiation pattern in the basilar to luminal direction. Real time imaging of the epithelium will be achieved by developing a vertical actuator that uses thin film piezoelectric materials that have the size, speed, force, and linearity to overcome motion artifact (>10 frames/sec). This device will be combined with a lateral scanning micro-mirror that is driven by parametric resonance. Affinity peptides will be selected using the technique of phage display by biopanning a high diversity library against cells and tissues that over express surface targets associated with neoplasia. Multiple probes can be near-infrared labeled to achieve the tissue penetration depths expected (>500 microns). Imaging performance will be validated by inserting the miniature prototype into the colon to evaluate the epithelium of the distal mucosa or by placing against the chest to image the epithelium of breast ducts of live animals. Public Health: This general purpose instrument can be used to study molecular processes in the epithelium of live animal models with sub-cellular resolution, high dynamic range, and full imaging depth, allowing for longitudinal investigation of mechanisms of cancer biology and providing a new platform for drug discovery.